A targeting construct containing the neomycin and HSTK genes and 10 kb of DNA genomic homology was constructed to disrupt the Muc5ac gene by homologous recombination. The construct was electroporated into mouse ES cells and 2 PCR positive clones of 170 clones analyzed were obtained. PCR and genomic Southern analysis verified targeting. The homozygous Muc5ac mutant mice have reduced level of mucus on the surface of the gastric mucosa and in the gastric pit. In addition, the middle ear of the Muc5ac-/- animals contain smaller goblet cells with less mucous compared to the wild type. The mononuclear inflammatory infiltrate found in the lamina propria of the middle ear of the Muc5ac-/- animals appears to be associated with the absence of Muc5ac, which makes the epithelium more susceptible to bacterial invasion. The conjunctiva and nasopharynx of the Muc5ac-/- mice also have significantly less and smaller goblet cells which contain decreased amount of mucous. Future investigations include gastritis and ulceration studies to examine the protective role of Muc5ac in stomach. In addition, we will use the murine model to examine the role Muc5ac plays in the pulmonary inflammation because MUC5AC is the principal gel-forming mucin upregulated in airway inflammation and its stimulated secretion is associated with airflow obstruction in asthma.